The sensitivity, resolving power, and speed of modern mass spectrometers now afford the opportunity to develop bottom-up footprinting methods capable of resolving significant structural and dynamics questions of membrane proteins. This bottom-up approach is a fundamentally more powerful alternative to the top-down mass spectrometry (MS) studies that have been mainly limited to bacterial membrane proteins. We focus on human proteins because they participate in almost all physiological processes and represent more than 60% of drug targets. They, however, represent the most challenging targets for traditional high-resolution structural methods. Structures of about 100 of these proteins are known to date, leaving a large gap for footprinting MS to fill. Our long-term goal is to develop comprehensive footprinting MS methods that offer a unique approach to structure and dynamics of membrane proteins in live cells and in vitro lipid bilayers. Our objective here is to synthesize new chemical probes that provide high footprinting coverage to reveal the ligand interaction and dynamic transport motion of ferroportin, a model protein representing the largest superfamily of membrane transporters and maintaining iron homeostasis in humans. Our hypotheses are: (1) Complementary chemistry can maximize the coverage of footprinting and thereby improve its spatial resolution. Furthermore, tuning the physical properties of the labeling reagents will allow access to the hydrophobic region of membrane proteins. (2) Photo- activated fast footprinting can reveal dynamic transporter motions taking place within milliseconds, which is beyond the current scope of membrane structure biology. (3) Bio-orthogonal irreversible labeling can be optimized to reveal the cellular structure state of membrane proteins, a structure that is elusive by crystallography or cryo-EM. Use of these conventional methods requires purified proteins, but most membrane proteins are insufficiently stable to withstand demanding purification. Live-cell footprinting completely avoids this giant difficulty. Our hypotheses are built on extensive preliminary data produced in our laboratories. Specifically, we continue to demonstrate our capability to explore new chemistry and synthesize new reagents. Our ongoing studies prove the principle that MS footprinting can reveal ligand-binding interaction of human membrane proteins in lipid bilayer, and can report on their native structural state and motion in live cells. To accomplish our goals, we will pursue three specific aims: (1) develop new chemical probes to provide high footprinting coverage of membrane proteins; (2) implement the new probes in lipid membrane systems to study the ligand interaction and millisecond motion of ferroportin; and (3) demonstrate the new probes' compatibility with live-cell footprinting by the detection of cellular motions and ligand interactions of ferroportin. Our innovative footprinting coupled with bottom-up MS proteomics analysis will establish effective, broad-based footprinting in live cells and lipid membranes. The significance of the proposed approach will expand because MS-based footprinting can be broadly applied by structural proteomics researchers to biomedically important human membrane proteins.